Influenza, commonly known as “the flu”, is an infectious respiratory disease caused by infection of human or zoonotic influenza viruses. Outbreaks of influenza occur annually during the cold months of each year, commonly known as “flu season”. These annual epidemics cause 3-5 million cases of severe illness and up to 500,000 deaths worldwide, although most symptoms of influenza infections are mild (World Health Organization: Influenza (Seasonal) on the World Wide Web at who.int/mediacentre/factsheets/fs211/en/). Each century, there are several influenza pandemics when influenza viruses infect a large proportion of human population and inflict significant morbidity and mortality around the world. The 1918 Spanish flu pandemic that killed 3 to 5 percent of the world's population is the most deadly pandemic in recorded history.
Current Strategies to Elicit Stem Domain Reactive Antibodies
Elicitation of broadly neutralizing antibodies (bNAbs) against all influenza viruses has been the ultimate goal of flu vaccine design. Majority of the isolated bNAbs recognize conserved epitopes in the HA stem domain. However, antibodies elicited by natural influenza virus infection or current flu vaccines mostly recognize the antigenic sites that surround the receptor binding site in the HA globular head domain due to the immunodominance of these antigenic sites. These antibodies are generally strain-specific as a result of the high variability of these antigenic sites among influenza virus strains. Thus current flu vaccines do not induce universal immunity against flu viruses.
Hemagglutinin (HA) is an integral membrane glycoprotein. It is the most abundant protein in influenza virus lipid bilayer envelope. About 500 molecules of HA are estimated on the surface of a virion. HA is the only protein required for adsorption and penetration of virus into host cells. It binds to host cell receptors and enables fusion between the virion lipid envelope and the host cell membrane. Through its ability to bind host cell receptors, HA determines the host range of an influenza strain.
HA is synthesized as a single chain precursor named HA0 in the endoplasmic reticulum (Stevens, J., et al., Science (2006) 312:404-410). The HA0 precursor has a signal peptide at its N-terminus and a membrane anchor sequence at its C-terminus. The N-terminal signal peptide is removed during the process when HA is transported across and anchored into the host cell membrane. HA is assembled into trimers of identical subunits in the endoplasmic reticulum and is then exported to the cell surface via the Golgi network. HA0 is cleaved at the C-terminal end of the maturation cleavage site (MCS) by specific host trypsin-like protease and converts to the mature form consisting of two disulfide-bond linked polypeptides, HA1 and HA2. HA1 is the larger N-terminal portion and HA2 is the smaller C-terminal portion of HA0. HA2 has the transmembrane region with a short C-terminal intracellular tail and anchors the HA to the membrane. Each mature HA has a total molecular weight of about 70 kDa with HA1 about 45 kDa and HA2 about 25 kDa. The atomic structures of the extracellular portion of HAs from many influenza strains have been determined. The structural model of a mature HA is shown in FIG. 1. All HAs, either as HA0 or the mature form, have the same overall trimeric structure with a globular head on a stem. Each HA monomer is anchored on membrane by a single C-terminal transmembrane region with a short hydrophilic cytoplasmic tail.
The globular head domain is made entirely of the HA1 and contains immunodominant epitopes. The stem domain is mostly made of the HA2 and contains conserved regions that are subdominant immunogenically. The globular head domain has an eight-stranded β-sheet structure at its core with surface loops and helices. The membrane proximal stem domain is composed of left-handed superhelix of a triple coiled-coil structure of residues from both HA1 and HA2. Each HA monomer has multiple glycosylation sites with a total carbohydrate of about 13 kDa of molecular weight or 19% of the total HA molecular weight. Most glycosylation is in the stem domain near the membrane surface. HA is also modified by palmitoylation of cysteine residues on the cytoplasmic tail (Veit, M., et al., J. Virol (1991) 65:2491-2500).
To circumvent the immunodominance of the globular head domain, immunogens have been designed based on the HA stem domain without the globular head domain (headless HAs). These attempts have been largely unsuccessful due to the difficulty of production of such molecules with proper tertiary structure (Krammer, F. and Palese, P. Curr Opin Virol (2013) 3:521-530; Eckert, D. M., and Kay, M. S., PNAS (2010) 107:13563-13564). HA2 by itself has been expressed in E. coli in soluble form that folds into its most stable post-fusion low-pH-induced conformation (Chen, J., et al., PNAS (1995) 92:12205-12209). By incorporating designed mutations to destabilize the low pH conformation of HA2, another HA2 construct (HA6) based on H3 HA was expressed in E. coli and refolded into the desired neutral pH pre-fusion conformation (Bommakanti, G., et al., PNAS (2010) 107:13701-13706). HA6 was highly immunogenic in mice and protected mice against the infection by homologous influenza A viruses. However, sera from HA6 immunized mice failed to neutralize virus in vitro, which could be due to limitation of the assay that only detect virus-neutralizing activity of the antibodies recognizing the globular head domain. Another “headless” HA stem domain construct with the HA2 and the region of HA1 in the stem domain has been described (Steel, J., et al., mBio (2010) 1:e00018-10). This immunogen protected mice against homologous influenza challenge and elicited antisera cross-reactive to heterologous HAs from within the same group. As with HA6, the antisera did not show neutralizing activity in vitro. These designs are all based on protein minimization by eliminating immunodominant regions.
Recently, through structure-based rational design and reiterative bNAb selection of construct libraries, stable trimeric H1 HA stem-only immunogens (headless mini-HAs) were made (Impagliazzo, A., et al., Science (2015) 349:1301-1306; Yassing, H., M., et al., Nat Med (2015) 21:1065-1070; Patent application WO2014/191435_A1). These stem-only mini-HA immunogens elicited expected antibodies against HA stem domain. Mice and ferrets immunized with these mini-HA immunogens were protected from lethal challenge of highly pathogenic H5 virus. In addition, a mini-HA immunogen elicited H5 neutralizing antibodies in cynomolgus monkeys. These mini-HA immunogens were selected for their binding to specific bNAbs. The lengthy reiterative bNAb selection process needs to be repeated for a different type of bNAbs. Although conformations of the bNAb epitopes were preserved in these mini-HA immunogens, the overall structures were different from the stem domain of native HA. Due to these structural changes, it is unlikely these headless mini-HA immunogens can be assembled into influenza viruses or influenza virus-like particles (VLPs). In addition, these headless mini-HA immunogens do not have the receptor binding site for host cell binding.
Chimeric HAs have been designed to direct the host immune response to the stem domain. Most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the HA stem domain and globular head domain of multiple influenza strains (Wrammert, J., et al., J. Exp Med (2011) 208:181-193). Immunization with HA derived from H5N1 influenza strains (a group 1 HA) that is not circulating in humans substantially increases HA stem-specific responses to group 1 circulating seasonal strains (Ellebedy, A. H., et al., PNAS (2014) 111:13133-13138; Nachbagauer, R., et al., J Virol (2014) 88:13260-13268; Whittle, J. R. R., et al., J. Virol (2014) 88:4047-4057). After each emergence of pandemic influenza virus strains in 1957, 1968, and 2009, existing seasonal virus strains were replaced in the human population by the novel pandemic strains.
It is hypothesized that exposure to new pandemic influenza virus strains with divergent globular head domains lead to affinity matured memory responses to the conserved epitopes in the stem domains (Palese, P. and Wang, T. T., mBio (2011) 2:e00150-11). In support of this hypothesis, engineered recombinant chimeric HA of H6, H9, or H5 globular head domain and H1 stem domain generated high titer stem-specific neutralizing antibodies (Pica, N., et al., PNAS (2012) 109:2573-2578; Krammer, F., et al., J. Virol (2013) 87:6542-6550). In these cases, the globular head domain of H1 HA was replaced by the globular head domain of H6, H9, or H5 from the same group 1 HA to which most of the human population is naïve. These replacements were made by replacing the H1 HA sequence between cysteine 52 and cysteine 277 of HA1 with the corresponding sequence of H6, H9, or H5. Cysteine 52 and cysteine 277 form a disulfide bond in the hinge region between the globular head domain and the stem domain. Similar chimeric HAs were made with H3 stem domain to develop vaccines for group 2 influenza strains (Krammer, F., et al., J. Virol (2014) 88:2340-2343; Margine, I., et al., J. Virol (2013) 87:10435-10446). Immunizing animals with these chimeric HAs induced stem-specific antibodies with broad neutralizing activity against each group of viruses. Human population has preexisting immunity to circulating H1 (group 1), H3 (group 2), and influenza B virus strains. Vaccination with a chimeric HA boosted antibody levels against the stem domains that are common to HAs of the circulating strains and the chimeric HA. Only a primary response was induced against the novel globular head domain on the chimeric HA to which humans are naïve. Subsequent boost with a second chimeric HA that possesses the same stem domain but a different head domain further increased stem-specific antibody levels. The results suggest changing the host exposure to the immunodominant epitopes in the globular head domain can increase the broadly protective immune responses against the immunosubdominant epitopes in the stem domain.
The six-amino-acid loop of antigenic Site B of A/WSN/33(H1N1) hemagglutinin can be replaced by the homologous antigenic Site B residues of HAs from A/Japan/57(H2N2) and A/Hong Kong/8/68(H3N2) (Li, S., et al., J. Virol (1992) 66: 399-404). These replacements do not interfere with the receptor binding function of HA. Recombinant influenza viruses with these chimeric HAs were replicated in MDCK (Madin-Darby Canine Kidney Epithelial Cells) cell culture. Viruses with chimeric HAs of A/WSN/33(H1N1) and A/Hong Kong/8/68(H3N2) induced antibodies against both A/WSN/33(H1N1) and A/Hong Kong/8/68(H3N2). These results suggest that the immunodominant antigenic sites of a HA can be replaced by the homologous corresponding immunodominant antigenic sites of other HAs from different strains. These replacements change the antigenic specificity of the resulting chimeric HAs.
In the foregoing paragraphs, therefore, the immunodominant epitopes in one influenza strain were replaced by the homologous immunodominant epitopes from another strain.
Another strategy is to dampen the immunodominant epitopes and refocus the host immune response towards the stem domain. The immunodominant antigenic sites in the globular head domain can be shielded by introducing additional glycosylation sites for hyperglycosylation (Eggink, D., et al., J. Virol (2014) 88:699-704, Patent application US2014/0004149_A1). The hyperglycosylation in globular head domain did not change the binding affinity of stem-reactive antibodies. Immunization of mice with the hyperglycosylated HA induced high titers of stem-reactive antibodies and protection against morbidity and mortality upon challenge with distinct seasonal viruses. Patent application US2013/0315929_A1 disclosed another method to dampen the immunodominant epitopes in the globular head domain by replacing some residues of those epitopes with other amino acids with less likelihood of being part of an epitope. The results suggest shielding immunodominant epitopes in the globular head domain can increase the broadly protective immune responses against the immunosubdominant epitopes in the stem domain.
HA as Carrier to Present Foreign Epitopes
Influenza HA has been used as a carrier for epitopes of V3-loop of HIV-1 envelope protein. Insertion of immunodominant epitope peptides of 12 to 22 residues in length from HIV-1 envelope protein gp120 to the HA immunodominant antigenic site, either the Site A or Site B, produced chimeric HAs with individual HIV-1 epitope in the globular head domain. No residues of Site A and Site B were removed. The immunodominant HIV-1 epitopes were inserted into the sites. The chimeric HAs induced immune responses to the HIV-1 V3-loop epitope in animals (Kalyan, N. K., et al., Vaccine (1994) 12:753-760; U.S. Pat. No. 5,591,823_A; Li, S., et al., J. Virol (1993) 67:6659-6666). The immunogenicity of the inserted epitopes appeared to be enhanced by HA since a very low dose of a chimeric HA protein was sufficient to induce antibodies specific to the inserted epitope. These results suggest that immunodominant foreign epitopes from proteins other than HA can be inserted to the immunodominant antigenic sites of HA. These chimeric HA molecules with inserted foreign immunodominant epitopes can induce immune responses to the inserted foreign immunodominant epitopes.